High affinity anti-prostate stem cell antigen (psca) antibodies for cancer targeting and detection

ABSTRACT

The present invention provides novel high affinity antibodies and fragments thereof that bind to the cancer antigen PSCA. The antibodies of the present invention may be used for cancer diagnosis, prognosis, treatment, visualization, and the like. The Present invention also provides methods for the detection, visualization, and treatment of various cancers expressing PSCA.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/676,348 filed on Aug. 5, 2010 which is a 371 of PCT/US08/75291 filedSep. 4, 2008 which claims benefit of U.S. Provisional Application No.60/969,939 filed on Sep. 4, 2007, all of which are incorporated hereinby reference in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant NumberCA092131, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The sequence listing contained in the file named“008074-5011-US01_SEQUENCE_TXT”, created on Dec. 5, 2014 and having asize of 33.3 kilobytes, has been submitted electronically herewith viaEFS-Web, and the contents of the txt file are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Prostate stem cell antigen (PSCA) a cell surface glycoprotein expressedin normal human prostate and bladder is over-expressed in prostatecancers (40% of primary tumors and 60-100% of lymph node and bone marrowmetastases). It is also highly expressed in transitional carcinomas ofthe bladder and pancreatic carcinoma. 1G8, an anti-PSCA mouse monoclonalantibody specific for PSCA demonstrated anti-tumor targeting activity invivo (Gu Z, et al. Cancer Res. 2005; 65:9495). This antibody washumanized by grafting on a human framework (Trastuzumab) and named 2B3(Olafsen T, et al. J. Immunotherapy 2007; 30:396).

The invention addresses the need for agents that have appropriatepharmacodynamic properties to target and image tumors that express PSCA.There is a tremendous need in the field for effective agents to imagecancers with sensitivity and specificity, particularly early stagetumors or ones with early metastasis not imageable by traditional means.As PSCA is highly expressed by most prostate, bladder and pancreatictumors, it is an important target in the detection, diagnosis,prognosis, and treatment of these cancers. The current inventiondescribes an innovative constructs with optimal characteristics fortumor imaging and targeting. They may also be used for tumor targetingof gene therapy, radioactivity therapy, and may have therapeutic utilityby themselves.

BRIEF SUMMARY OF THE INVENTION

This invention describes engineered antibodies that recognize a novelcell surface marker in prostate and other cancers with high affinity.These genetically engineered antibody fragments are tailoredspecifically for in vivo use for targeting and detection.

The invention addresses the need for agents that have appropriatepharmacodynamic properties to target and image tumors that express PSCA.There is a tremendous need in the field for effective agents to imagecancers with sensitivity and specificity, particularly early stagetumors or ones with early metastasis not imageable by traditional means.PSCA is highly expressed by most prostate, bladder and pancreatic tumorsand is a promising target. The current invention describes an innovativemolecule with optimal characteristics for tumor imaging. It may also beuseful for tumor targeting of gene therapy, radioactivity or may havetherapeutic utility by itself.

There are multiple embodiments. For instance, there are a variety ofengineered antibody formats, such as scFvs, diabodies, triabodies,minibodies, and scFv-Fc. In general, the agent should at leastdemonstrate bivalent, as opposed to monovalent binding. The overallsize, shape, and domain composition of the agent can be varied to suitthe final application. Engineered fragments that exhibit optimaltargeting in humans may be slightly different from formats that areoptimal in mice. Since one goal is human application, the inventionincorporates a humanized set of antibody variable regions, as well ashuman hinge and constant regions. Additional embodiments would includefully human variable regions. The proteins can be expressed in a varietyof systems, including microbial, insect cells, mammalian cell cultureand transgenic animals.

For imaging purposes, a variety of radionuclides can be attached to theengineered antibodies for detection with gamma or SPECT cameras, or PETscanners. For therapy one can attach drugs, toxins, cytokines, enzymes,or other therapeutic moieties for PSCA-targeted delivery to tumors. Theengineered PSCA-specific antibodies can be coupled to nanosensors fordetection (in vitro or in vivo) or nanoparticles for delivery (in vivo).One can also incorporate the PSCA antibody fragments into viral vectorsfor targeted gene therapy of tumors.

The invention addresses the unmet need for imaging of cancer, in earlydiagnosis or diagnosis of metastatic disease. In particular, there is acritical need for better agents for imaging prostate cancer fordetection and staging. PSCA antibody fragment imaging will be veryuseful for imaging bone metastases and assessing response to treatment;there are no good imaging approaches currently available. Detection ofpancreatic cancer is a critical need, and an imaging agent would beuseful in high-risk patients. The invention describes high-affinity,highly specific engineered antibodies tailored for in vivo targeting anddetection of PSCA in prostate cancer, bladder cancer, and pancreaticcancer patients.

Accordingly, in a first aspect, the invention provides high affinityPSCA antigen binding protein constructs which can be used in thetreatment and detection of cancers which overexpress PSCA. In someembodiments, these constructs are minibodies, diabodies, triabodies,ScFv, or ScFv-Fc as described further below. In one embodiment, theinvention provides an antigen binding protein construct directed towarda manunalian PSCA protein (e.g., human, murine) selected from the groupconsisting of a minibody, a diabody, and scFv-Fc wherein the selectedconstruct has VL and V_(H) domains that are substantially identical,respectively, to the V_(L) domain and the V_(H) domain of an anti-PSCAantibody. For example, the construct can be a minibody in which theV_(L) and V_(H) chain variable domains of the anti-PSCA antibody arefused to part of the hinge region of an antibody, an amino acid linkerand the C_(H)3 domain of an immunoglobulin molecule. In otherembodiments, the construct is a diabody.

In embodiments, where the construct is a minibody or diabody or scFV-Fe,the anti-PSCA antibody can be a humanized antibody and the C_(H)3 domainis from a human immunoglobulin molecule. In preferred embodiments, theanti-PSCA antibody is 2B3 or 1G8. In yet other embodiments, theconstruct is a minibody having V_(H) and V_(L) domains substantiallyidentical to an scFv fragment designated herein as A11, A2, or C5. Instill further embodiments, the construct has a C_(H)3 domain is from ahuman immunoglobulin molecule. For instance, the construct may have ahinge region and C_(H)3 domain are the human IgG hinge region and humanC_(H)3 domain.

In some embodiments, the anti-PSCA antibody is a monoclonal antibodydesignated 1G8 (ATCC No. HB-12612), 2A2 (ATCC No. HB-1203), 2H9 (ATCCNo. HB-12614), 3C5 (ATCC No. HB-12616), 3E6 (ATCC No. HB12618), 3G3(ATCC No. HB-12615), or 4A10 (ATCC No. HB-12617).

The constructs according to the invention can also be linked totherapeutic agents or detectable markers. In some embodiments, thetherapeutic agent is a cytotoxic agent. For instance, the agent can bericin, ricin A-chain, doxorubicin, daunorubicin, TAXOL, ethiduimbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin Achain, alpha-sarcin, gelonin mitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, maytansinoids, or glucocorticoidricin. In other embodiments,the therapeutic agent is a radioactive isotope. The radioactive isotopecan be selected, for instance, from the group consisting of ²¹²Bi, ¹³¹I,¹¹¹In, 90Y and ¹⁸⁶Re. In other embodiments the construct is linked to ananti-cancer pro-drug activating enzyme capable of converting a pro-drugto its active form.

In another preferred embodiments of the above, the PSCA targeted by theanti-PSCA antibody is human PSCA.

In another aspect, the invention provides an antigen binding proteinconstruct selected from the group consisting of a minibody, a diabody,scFv and scFv-Fc wherein the selected construct has V_(L) and V_(H)domains that are substantially identical to the V_(L) and V_(H) domainsof 2B3 or an scFv variant designated herein as A11, A2, or C5. In otherembodiments, the binding constructs of the present invention maycomprise one or more mutations found in the variant antibodies A11, A2,or C5.

In yet another aspect, the present invention provides an antigen bindingprotein construct selected from the group consisting of a minibody, adiabody, scFv and scFv-Fc wherein the selected construct comprises CDRregions of an anti-PSCA antibody. In certain embodiments, the anti-PSCAantibody will bind to PSCA with an affinity equal to or greater than theantibody designated 2B3. In other embodiments, the anti-PSCA antibodymay be an affinity matured antibody, wherein the affinity maturedantibody comprises a higher affinity for PSCA than does the parentalantibody.

In another aspect, the anti-PSCA construct according to the invention islabeled with a detectable marker. The marker can be for instance, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. Manyradionuclides may be used as imaging labels, including withoutlimitation, ¹²⁴I, ⁸⁶Y, ¹⁸F, ⁹⁴Tc, and the like. One of skill in the artwill know of other radionuclides particularly well suited for use in thepresent invention.

In further other embodiments of any of the above, the invention providesa pharmaceutical composition of the constructs according to theinvention.

In another aspect still, the invention provides methods for treating asubject having cancer (e.g, prostate, pancreatic or bladder cancer), orinhibiting the growth of a prostate cancer cell expressing a ProstateStem Cell Antigen (PSCA) protein comprising contacting the cancer cell(e.g., prostate, bladder, pancreatic cancer cell, with a constructaccording to the invention in an amount effective to inhibit the growthof the cancer cell. The method can kill the cancer cell. In someembodiments, the construct recognizes and binds the PSCA protein asshown below beginning with leucine at amino acid position 22 and endingwith alanine at amino acid position 99. In additional embodiments, themethod further comprises administering to a chemotherapeutic drug,radiation therapy. In some embodiments, the subject is also treated withhormone ablation therapy or hormone antagonist therapy.

The treatments may be given to the patient or subject by intravenously,intraperitoneally, intramuscularly, intratumorally, or intradermally. Insome embodiments, the contacting comprises administering the constructdirectly into a prostate cancer, a bladder cancer, a pancreatic canceror a metastasis thereof.

In another aspect, the invention provides methods of detecting acancerous cell in a subject by contacting the cancer cell with aconstruct which bears a detectable marker. The methods can be used inscreening patients at increased risk of cancer or to monitory responseto therapy or to develop a prognosis for the cancer (e.g., prostate,bladder, or pancreatic cancers. The methods are particularlyadvantageous in detecting metastases of the cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic overview of anti-PSCA minibody. A gene encoding theminibody is assembled in the order V_(L)-linker-V_(H-)hinge-C_(H)3, withthe hinge and C_(H)3 domains derived from human IgGl. The protein selfassembles into 80-kDa dimers.

FIG. 2: Comparison of 2B3 ScFv variant protein sequences. The V_(L)sequence and part of the linker are in the top frame, the rest of thelinker and V_(H) sequences are in the lower frame. Also indicated arethe CDRs: underlined wild type sequence. The mutations are highlightedin different shading. The parental ScFv P-2B3 sequence (SEQ ID NO:4) andvariant C5 (SEQ ID NO:5), A2 (SEQ ID NO:6), and A11 (SEQ ID NO:7) ScFvsequences are shown.

FIG. 3: DNA sequence (SEQ ID N0:8) and translated protein sequence ofthe parental 2B3 minibody (SEQ ID N0:9). Also indicated are the startingpoints of the following protein segments: signal peptide (for mammaliansecretion), light chain variable region (V_(L)), the 15 amino acidinterdomain peptide linker, heavy chain variable region (V_(H)), humanIgGl hinge sequence and 10 amino acid extension, and the human IgGl CHjdomain followed by two stop codons.

FIG. 4: SDS-PAGE analysis of the four minibodies in non-reducingconditions: parental (p), A11, A2 and C5. The arrow indicates theexpected molecular weight of a minibody.

FIG. 5: Size-exclusion HPLC of A11 minibody showing homogenous peak atexpected molecular size.

FIG. 6A-FIG. 6B: Affinity ranking (A) by Competitive ELISA bindingassay: Plates were coated with CEA and biotinylated intact, chimericanti-CEA antibody was used as probe. (B) by flow cytometry: 5 pg/ml ofeach minibody was incubated with PSCA expressing cells. Cells were thenstained with anti Human Fe PE conjugated.

FIG. 7A-FIG. 7F: Co-registered microPET/microCT scan of a nude mousebearing LAPC-9AD (PSCA-positive human prostate cancer) xenografts. Themouse was injected with 1-1 24 radiolabeled A11 minibody variant andscanned serially. A, B, C; saggital sections; D, E, F; coronal sections.A, D; coregistered microPET and microCT, showing ROI (region ofinterest) as a white rectangle. B, E; microPET images and ROI C, F;microPET images only.

FIG. 8: Biodistribution and microPET Imaging ranking of the 2B3 minibodyvariants. Biodistribution unit is a % ID/g values of weighed tissues iny-counter after 21 or 25 hours injection time. MicroPET Imaging valueswere obtained on the average values of 4 RO is as shown in FIG. 6 anddescribed in the materials and methods.

FIG. 9: Summary of PSCA data and properties.

FIG. 10: Depictions of various constructs according to the invention.

FIG. 11: Schema for producing 2B3 variants.

FIG. 12: MicroPET imaging and biodistribution data for variousconstructs.

FIG. 13: MicroPET imaging and biodistribution results for A11 and parent2B3.

FIG. 14: MicroPET imaging and biodistribution data for pancreatic cancerCapan-1 xenographic mice using parental 2B3 and variant A11 anti-PSCAminibodies.

FIG. 15: MicroPET imaging and biodistribution data for pancreatic cancerMIA PaCa-2 xenographic mice using parental 2B3 and variant A11 anti-PSCAminibodies.

FIG. 16A-FIG. 16B Amino acid sequences of parental (A) 2B3 (SEQ ID NO:10) and variant A2 (SEQ ID NO: 11), A11 (SEQ ID NO: 12), (B) and C5 (SEQID NO: 13) anti-PSCA minibodies.

DETAILED DESCRIPTION OF THE INVENTION

Prolonged clearance kinetics have hampered the development of intactantibodies as imaging and therapeutic agents, despite their ability toeffectively deliver radionuclides to tumor targets in vivo. Here, wealso report genetically engineered antibody fragments which displayrapid, high-level tumor uptake coupled with rapid clearance from thecirculation in a nude mouse model to allow ready detection of tumors invivo.

The 2B3 antibody was humanized by grafting on a human framework(Trastuzumab) and named (Olafsen T, et al. J. Immunotherapy 2007;30:396; which is incorporated by reference in its entirety with respectto the description, biological activity, and making of the antibody).Antibody fragments of 2B3 have been generated for PET imagingapplication. One of these antibody fragments, the 2B3 minibody hasdemonstrated rapid and specific localization to PSCA-expressing tumorsin a murine model. However, humanization has decreased the affinity ofthe intact antibody for PSCA from a Kd of 2.6 nM to 16.7 nM. Inaddition, reformatting of intact antibody into antibody fragments canalso affect the binding efficiency. It has been shown that quantitativetumor retention of ScFvs increases with affinity but only to a thresholdclose to 10⁻⁹M (Adams G P, et al. Cancer Res. 2001; 61:4750). There areno published data on the effect of minibody affinity on tumor targetingand imaging properties. Here, we describe the generation of three highaffinity anti-PSCA ScFvs, and the subsequent generation andcharacterization of three high affinity anti-PSCA minibodies. Wegenerated minibody fragments with better tumor targeting/imagingaptitude. In some embodiments, with the regard to the constructs of theinvention, there is a proviso that the construct comprises a V_(H) orV_(L) domain that is not identical to a corresponding domain or the 2B3antibody.

In one embodiment, the present invention provides antigen bindingconstructs selected from the group consisting of an antibody, aminibody, a diabody, an scFv, an scFv-Fe, and the like, wherein the VLand VH domains are substantially identical to those found in 2B3. Inasecond embodiment, the antigen binding construct may comprise one ormore mutations found in an antibody variant designated herein as A11,A2, or C5. Ina third embodiment, the binding construct comprises atleast one mutation at a residue corresponding to an amino acid of SEQ IDNO:4 selected from the group consisting of T5, S10, VI5, S91, S123,S131, N179, T182, I194, A203, G213, Q228, and a combination thereof. Incertain embodiments, the at least one mutation comprises a mutationcorresponding to a mutation in SEQ ID NO:4 selected from the groupcomprising T5I, SI0I, VI5M, S9IG, ΔI23, S13I Y, N179Y, TI825, I194M,A203V, E213K, Q228R, and a combination thereof. In other embodiments,the binding constructs of the invention comprise mutations correspondingto those found in variants A11, A2, or C5.

In a certain embodiment, the present invention provides a minibodywherein the V_(L) and V_(H) domains are substantially identical to thosefound in SEQ ID NO:10. In one embodiment, a minibody of the presentinvention comprises an amino acid sequence of SEQ ID NO:10. In otherembodiments, the minibody comprises an amino acid sequence that issubstantially identical to SEQ ID NO:10, wherein said minibody binds toPSCA with a higher affinity than a minibody of SEQ ID NO:10. In yetother embodiments, the minibody may comprise one or more mutations at aresidue corresponding to an amino acid of SEQ ID N0:10 selected from thegroup comprising T5, S10, V15, S91, S123, S131, N179, TI82, I194, A203,G213, Q228, and a combination thereof. In yet another embodiment, theconstruct may comprise a sequence selected from SEQ ID NOS:11, 12, and13.

In one embodiment, the invention provides an antigen binding proteinconstruct selected from the group consisting of a minibody, a diabody,scFv and scFv-Fc wherein the selected construct comprises CDR regions ofan anti-PSCA antibody. In one embodiment, the binding protein constructwill comprise at least one CDR region selected from a CDR-L 1, CDR-12,CDR-13, CDR-HI, CDR-H2, or CDR-H3 from an anti-PSCA antibody. In yetother embodiments, the protein binding construct may comprise all 3light chain CDR regions or all three heavy chain CDR regions. In oneembodiment, the protein binding constructs of the present invention maycomprise all of the CDR regions of an anti-PSCA antibody. In certainembodiments, the anti-PSCA antibody will bind to PSCA with an affinityequal to or greater than the antibody designated 2B3. Motherembodiments, the anti-PSCA antibody may be an affinity matured antibody,wherein the affinity matured antibody comprises a higher affinity forPSCA than does the parental antibody. In particular embodiments, theparental anti-PSCA antibody may be selected from the group consisting of1G8 (ATCC No. HB-12612), 2A2 (ATCC No. HB-1203), 2H9 (ATCC No.HB-12614), 3C5 (ATCC No. HB-12616), 3E6 (ATCC No. HB12618), 3G3 (ATCCNo. HB-12615), 4A10 (ATCC No. HB-12617), and 2B3. In other embodiments,the CDRs may be selected from those found in SEQ ID NOS:10, 11, 12, and13.

A “minibody” is an engineered antibody construct comprised of thevariable heavy (VH) and variable light (VL) chain domains of a nativeantibody fused to the hinge region and to the CH3 domain of theimmunoglobulin molecule (see, FIG. 1). Minibodies are thus smallversions of whole antibodies encoded in a single protein chain whichretain the antigen binding region, and the CH3 domain which to permitassembly into a bivalent molecule and the antibody hinge to accommodatedimerization by disulfide linkages. In contrast, native antibodies arecomprised of four chains, two heavy and two light. The size, valency andaffinity of the minibody is particularly suited for in vivo targeting.Expression in bacterial or mammalian cells is simplified becauseminibodies can be produced as single amino acid chains (see, U.S. Pat.No. 5,837,821) which is incorporated by reference herein in its entiretyand particularly with reference to minibodies, their structure, ways ofmaking them, and their suitable pharmaceutical formulations.

A “diabody” comprises a first polypeptide chain which comprises a heavy(VH) chain variable domain connected to a light chain variable domain(VL) on the first polypeptide chain (VH-VL) connected by a peptidelinker that is too short to allow pairing between the two domains on thefirst polypeptide chain and a second polypeptide chain comprising alight chain variable domain (V_(L)) linked to a heavy chain variabledomain V_(H) on the second polypeptide chain (VL-VH) connected by apeptide linker that is too short to allow pairing between the twodomains on the second polypeptide chain. The short linkages force chainpairing between the complementary domains of the first and the secondpolypeptide chains and promotes the assembly of a dimeric molecule withtwo functional antigen binding sites.

To construct bispecific diabodies the V-domains of different antibodies(e.g., antibody A and antibody B) are fused to create the two chains(e.g., VHA-VLB, VHB-VLA). Each chain is inactive in binding to antigen,but recreates the functional antigen binding sites of antibodies A and Bon pairing with the other chain.

PSCA and its expression in cancer of the prostate, bladder, and pancreasis disclosed in U.S. Pat. No. 6,756,036 which is incorporated byreference in its entirety. The human PSCA translated amino acid sequenceis:

(SEQ ID NO: 1) MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQOYYVGKKNITCCDTDLCNASGAHALQPAAAILAllPAL GLLUJGPGQL

The terms “substantially identical” in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same (i.e., about 80%identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) over thereferenced sequences or portions, when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site or the like). The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. Preferably, the sequence identity is atleast 85%, 90%, 95% 97% between two referenced domains. In someembodiments, the difference in sequence is just by one, two, three orfour, or from five to 12, amino acids as to referenced sequence ordomain. As described below, the preferred algorithms can account forgaps and the like. Preferably, identity exists over a region that is atleast about 15 amino acids or nucleotides in length, or more preferablyover a region that is 15-50 amino acids or nucleotides in length. Inother embodiments, the identity may exist over a region that is at leastabout 50, 100, 150, 200, or more amino acids.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected over aspecified range of residues (e.g., 20 to 50, usually about 50 to about200, more usually about 100 to about 150) in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, JMol. Bioi. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J Mol. Bioi. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. Tis referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0) and N (penalty score for mismatching residues;always<0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Nat!. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A “label” or a “detectable moiety” or “detectable marker” is acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, lgM, lgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to VwCH1 by a disulfide bond.The F(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty eta!., Nature 348:552-554(1990)).

For preparation of suitable antibodies or constructs of the inventionand for use according to the invention, e.g., recombinant, monoclonal,or polyclonal antibodies, many techniques known in the art can be used(see, U.S. Patent Application Publication No. 20070196274 and U.S.Patent Application Publication No. 20050163782, which are eachincorporated by reference in their entireties, particularly with respectto minibody and diabody design) (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991);Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding,Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genesencoding the heavy and light chains of an antibody of interest can becloned from a cell, e.g., the genes encoding a monoclonal antibody canbe cloned from a hybridoma and used to produce a recombinant monoclonalantibody. Gene libraries encoding heavy and light chains of monoclonalantibodies can also be made from hybridoma or plasma cells. Randomcombinations of the heavy and light chain gene products generate a largepool of antibodies with different antigenic specificity (see, e.g.,Kuby, Immunology (3rd ed. 1997)). Techniques for the production ofsingle chain antibodies or recombinant antibodies (U.S. Pat. No.4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce antibodiesto polypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanized orhuman antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks eta!., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); andLonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see. e.g., U.S. Pat. No. 4,676,980, WO 91100360; WO92/200373; and EP 03089).

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody. For example, the antibody derivatives include, withoutlimitation, antibodies that have been modified by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, and the like. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-natural amino acids.

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Bioi. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. The preferred antibodies of, and for useaccording to the invention include humanized and/or chimeric monoclonalantibodies.

In some embodiments, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein. Such effector moieties include but are not limited to, ananti-tumor drug, a toxin, a radioactive agent, a cytokine, a secondantibody or an enzyme. Further, the invention provides an embodimentwherein the antibody of the invention is linked to an enzyme thatconverts a prodrug into a cytotoxic agent.

The immunoconjugate can be used for targeting the effector moiety to aPSCA-positive cell, particularly cells, which overexpress the PCSAprotein. Such differences can be readily apparent when viewing the bandsof gels with approximately similarly loaded with test and controlssamples. Examples of cytotoxic agents include, but are not limited toricin, doxorubicin, daunorubicin, TAXOL, ethiduim bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin(PE) A, PE40, abrin, and glucocorticoid and other chemotherapeuticagents, as well as radioisotopes. Suitable detectable markers include,but are not limited to, a radioisotope, a fluorescent compound, abioluminescent compound, chemiluminescent compound, a metal chelator oran enzyme.

In some embodiments, the invention provides antigen binding proteinconstructs used to systemically to treat cancer (e.g., prostate,pancreatic or bladder cancer) alone or when conjugated with an effectormoiety. PSCA-targeting constructs conjugated with toxic agents, such asricin, as well as unconjugated antibodies can be useful therapeuticagents naturally targeted to PSCA bearing cancer cells. Such constructscan be useful in blocking invasiveness.

Additionally, the antigen-binding protein constructs of the inventioncan be used to treat cancer. In such a situation, the construct isjoined to at least a functionally active portion of a second protein ortoxic molecule having therapeutic activity. The second protein caninclude, but is not limited to, an enzyme, lymphokine, oncostatin ortoxin. Suitable toxins include doxorubicin, daunorubicin, TAXOL,ethiduim bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, ricin, abrin,glucocorticoid and radioisotopes.

Techniques for conjugating therapeutic agents to constructs according tothe invention are well known (see, e.g., Amon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985) Hellstrom et al., “Antibodies For DrugDelivery” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical, Applications, Pinchera et al. (eds.), pp.475-506 (1985) and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, hnmunol. Rev., 62:119-58(1982)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide or construct according to the invention, refers to abinding reaction that is determinative of the presence of the protein,often in a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein at least two times the background and moretypically more than 10 to 100 times background. Specific binding to anantibody under such conditions requires a construct be selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select constructs specifically immunoreactive with PSCA.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Using Antibodies, A Laboratory Manual (1998) for a descriptionof immunoassay formats and conditions that can be used to determinespecific immunoreactivity).

By “therapeutically effective dose or amount” herein is meant a dosethat produces effects for which it is administered. The exact dose andformulation will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro,Editor (2003), and Pickar, Dosage Calculations (1999)).

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, e.g., Berge eta/.,Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Other pharmaceutically acceptable carriersknown to those of skill in the art are suitable for the presentinvention.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

The methods fmd particular application in the diagnosis, prognosis andtreatment of cancers which overexpress PSCA, for example, prostate,pancreatic and bladder cancers. In certain embodiments the methods areapplied to hormone refractory or therapy resistant cancers. In certainembodiments the methods are applied to metastatic cancers.

Treatment will generally involve the repeated administration of theconstructs and their immunoconjugates via an acceptable route ofadministration such as intravenous injection (N), at an effective dose.Dosages will depend upon various factors generally appreciated by thoseof skill in the art, including without limitation the type of cancer andthe severity, grade, or stage of the cancer, the binding affinity andhalf life of the agents used, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic agents used in combination with the treatment method ofthe invention. Typical daily doses may range from about 0.1 to 100mglkg. Doses in the range of 10-500 mg of the constructs or theirimmunoconjugates per week may be effective and well tolerated, althougheven higher weekly doses may be appropriate and/or well tolerated. Theprincipal determining factor in defining the appropriate dose is theamount of a particular agent necessary to be therapeutically effectivein a particular context. Repeated administrations may be required inorder to achieve tumor inhibition or regression. Initial loading dosesmay be higher. The initial loading dose may be administered as aninfusion. Periodic maintenance doses may be administered similarly,provided the initial dose is well tolerated.

Direct administration of the constructs is also possible and may haveadvantages in certain contexts. For example, for the treatment ofbladder carcinoma, the agents may be injected directly into the bladder.

In another embodiment, the invention provides polynucleotides comprisinga nucleotide sequence encoding an antibody of the invention andfragments thereof. In one embodiment, the present invention provides anexpression vector encoding for an antibody or fragment thereof of thepresent invention. In another embodiment, the present invention providespolynucleotides encoding an antibody of the present invention for use ingene therapy or in vivo administration.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-0-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J Bioi. Chern. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, eDNA, mRNA,oligonucleotide, and polynucleotide.

Methods of Administration and Formulation

The constructs are administered to a subject in accord with knownmethods, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous or subcutaneous administration is preferred. Theadministration may be local or systemic.

The compositions for administration will commonly comprise an agent asdescribed herein dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration will vary according to the agent. Actual methods forpreparing parenterally administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

The pharmaceutical compositions can be administered in a variety of wlit dosage forms depending upon the method of administration. Forexample, unit dosage forms suitable for oral administration include, butare not limited to, powder, tablets, pills, capsules and lozenges. It isrecognized that constructs when administered orally, should be protectedfrom digestion. This is typically accomplished either by complexing themolecules with a composition to render them resistant to acidic andenzymatic hydrolysis, or by packaging the molecules in an appropriatelyresistant carrier, such as a liposome or a protection barrier. Means ofprotecting agents from digestion are well known in the art.

Pharmaceutical formulations, particularly, constructs andimmunoconjugates and inhibitors for use with the present invention canbe prepared by mixing a construct having the desired degree of puritywith optional pharmaceutically acceptable carriers, excipients orstabilizers. Such formulations can be lyophilized formulations oraqueous solutions. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations used.Acceptable carriers, excipients or stabilizers can be acetate,phosphate, citrate, and other organic acids; antioxidants (e.g.,ascorbic acid) preservatives low molecular weight polypeptides;proteins, such as serum albumin or gelatin, or hydrophilic polymers suchas polyvinylpyllolidone; and amino acids, monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents; and ionic and non-ionic surfactants (e.g.,polysorbate); salt-forming counter-ions such as sodium; metal complexes(e. g. Zn-protein complexes); and/or non-ionic surfactants. Theconstruct can be formulated at a concentration of between 0.5-200 mg/ml,or between 10-50 mg/ml.

The formulation may also provide additional active compounds, including,chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitoryagent, and anti-hormonal agent. The active ingredients may also preparedas sustained-release preparations (e.g., semi-permeable matrices ofsolid hydrophobic polymers (e.g., polyesters, hydrogels (for example,poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)),polylactides. The antibodies and immunoconjugates may also be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions.

The compositions can be administered for therapeutic or prophylactictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease (e.g., cancer) in a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. A “patient” or “subject” for the purposesof the present invention includes both humans and other animals,particularly mammals. Thus the methods are applicable to both humantherapy and veterinary applications. In the preferred embodiment thepatient is a mammal, preferably a primate, and in the most preferredembodiment the patient is human. Other known cancer therapies can beused in combination with the methods of the invention. For example, thecompositions for use according to the invention may also be used totarget or sensitize a cell to other cancer therapeutic agents such as5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.

In other embodiments, the methods of the invention may be practicedtogether with other cancer therapies (e.g, radical prostatectomy),radiation therapy (external beam or brachytherapy), hormone therapy(e.g., orchiectomy, LHRH-analog therapy to suppress testosteroneproduction, anti-androgen therapy), or chemotherapy. Radicalprostatectomy involves removal of the entire prostate gland plus somesurrounding tissue. This treatment is used commonly when the cancer isthought not to have spread beyond the tissue. Radiation therapy iscommonly used to treat prostate cancer that is still confined to theprostate gland, or has spread to nearby tissue. If the disease is moreadvanced, radiation may be used to reduce the size of the tumor. Hormonetherapy is often used for patients whose prostate cancer has spreadbeyond the prostate or has recurred. The objective of hormone therapy isto lower levels of the male hormones, androgens and thereby cause theprostate cancer to shrink or grow more slowly. Luteinizinghormone-releasing hormone (LHRH) agonists decrease the production oftestosterone. These agents may be injected either monthly or longer. Twosuch analogs are leuprolide and goserelin. Anti-androgens (e.g.,flutamide, bicalutamide, and nilutamide) may also be used. Totalandrogen blockade refers to the use of anti-androgens in combinationwith orchiectomy or LHRH analogs, the s combination is called.Chemotherapy is an option for patients whose prostate cancer has spreadoutside of the prostate gland and for whom hormone therapy has failed.It is not expected to destroy all of the cancer cells, but it may slowtumor growth and reduce pain. Some of the chemotherapy drugs used intreating prostate cancer that has returned or continued to grow andspread after treatment with hormonal therapy include doxorubicin(Adriamycin), estramustine, etoposide, mitoxantrone, vinblastine, andpaclitaxel. Two or more drugs are often given together to reduce thelikelihood of the cancer cells becoming resistant to chemotherapy. Smallcell carcinoma is a rare type of prostate cancer that is more likely torespond to chemotherapy than to hormonal therapy.

The combined administrations contemplates co-administration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by intravenous infusion, orally,topically, intraperitoneally, intravesically or intrathecally.Parenteral administration, oral administration, and intravenousadministration are the preferred methods of administration. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents.

Preferred pharmaceutical preparations deliver one or more constructsaccording to the invention, optionally in combination with one or morechemotherapeutic agents or immunotherapeutic agents, in a sustainedrelease formulation. The construct may be administered therapeuticallyas a sensitizing agent that increases the susceptibility of tumor cellsto other cytotoxic cancer therapies, including chemotherapy, radiationtherapy, immunotherapy and hormonal therapy.

In therapeutic use for the treatment of cancer, the constructs utilizedin the pharmaceutical method of the invention are administered at theinitial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A dailydose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg toabout 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kgto about 50 mg/kg, can be used. The dosages, however, may be varieddepending upon the requirements of the patient, the severity of thecondition being treated, and the compound being employed. For example,dosages can be empirically determined considering the type and stage ofcancer diagnosed in a particular patient. The dose administered to apatient, in the context of the present invention should be sufficient toeffect a beneficial therapeutic response in the patient over time.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired.

The pharmaceutical preparations for use according to the invention aretypically delivered to a mammal, including humans and non-human mammals.Non-human mammals treated using the present methods include domesticatedanimals (i.e., canine, feline, murine, rodentia, and lagomorpha) andagricultural animals (bovine, equine, ovine, porcine).

Methods of Tumor Imaging

In certain embodiments, the present invention provides methods ofimaging cancer cells or tumors in vivo through administration ofantibodies of the invention. In one embodiment, the present inventionprovides a method of imaging a cancer cell in vivo, the methodcomprising administering a labeled anti-PSCA antibody to a mammal andimaging the antibody in vivo. The methods of the present invention maybe used to image a cancer cell in mammal, including without limitation,a mouse, rat, hamster, rabbit, pig, human, and the like.

Methods of in vivo imaging are well !mown in the art and include withoutlimitation, magnetic resonance imaging (MRI), nuclear magnetic resonance(NMR) (R. Weissleder, 1999, Radiology 212:609-14), computerized axialtomography (CAT) scan, cooled charged coupled device (CCD) cameraoptical imaging (Honigman, et al., 2001 Mol. Ther. 4:239-249),bioluminescent optical imaging (P R Contag, et al., 1998 Nat. Med.4:245-247), position emission tomography (PET) (ME Phelps, 1991Neurochemical Research 16:929-994; J G Tjuvajev, et al., 1998 Cancer Res58:4333-4341), single photon emission computed tomography (J G.Tjuvajev, et al., 1996 Cancer Res. 45:4087-4095), microPET (reviewed inMcVeigh, 2006, Circ. Res. 98:879-86), and the like.

EXAMPLES

The following examples are offered to illustrate, but not limit, theclaimed invention.

Example 1

This example describes the construction of a mutant scFv yeast displaylibrary and the selection of ScFv mutants with improved PSCA bindingaffinity.

Oligonucleotides and vectors used in the construction of the libraryinclude; Gap 5′:

5′-TTAAGCTTCTGCAGGCTAGTG-3′ (SEQ ID NO:2); Gap 3′:5′-GAGACCGAGGAGAGGGTTAGG-3′ (SEQ ID NO:3); pYD2 inside the Ncoi-Notlrestriction sites (Razai A, et al. J Mol Bioi. 2005; 35 1:1 58).

2B3 ScFv gene was first cloned into the yeast display vector pYD2 (RazaiA, et al. J Mol. Bioi. 2005; 351:158) using Ncol-Notl restriction sites.A bacterial clone with the correct sequence was amplified and DNAextracted with QIAprep Spin Miniprep. Random mutations were introducedinto the 2B3 Scfv gene using error prone PCR as follows: 2B3 ScFv genewas subject to 20 cycles of PCR with Taq in the presence of IOO mM MnC12to generate random mutations. The PCR product was run on an agarose geland purified using QIAquick gel extraction. The purified PCR product wasre-amplified using a proof reading DNA polymerase for 35 cycles. BothPCRs were carried out with the GapS' and Gap3′ primers. The amplified2B3 Fv gene was again run on an agarose gel and purified using QIAquickgel extraction. Mutated scFv genes and NcoI-Notl digested pYD2 were usedto transform LiAc-treated EBY 100 cells by gap repair. The resultinggene repertoire was cloned into pYD2 using gap repair to create alibrary of 5.9×10⁵ transformants Transformation mixes were cultured andsub-cultured in SD-CAA. Library size was determined by plating serialdilutions of the transformation mixture on SD-CAA plates.

For selection, scFv display was induced by culturing in SG-CAA mediaplus zeocin for 24 hours at 20° C. For the first round of selection 20million yeast (more than 30 times the library size) were washed andresuspended in FACS buffer (phosphate-buffered saline (Ph 7.4), 0.5%bovine serum albumin) to which 200 nM of PSCA human Gamma 1 fusionprotein was added and incubated for 1 hour at room temperature. Theconcentrations of PSCA human Gamma 1 used for round 2, 3 and 4 ofsorting were 5 nM, 2 nM and 1 nM respectively. Cells were incubated for30 minutes with secondary antibodies at 4° C., washed once with FACSbuffer, resuspended in 200-500 μl of FACS buffer and sorted on aFACSAria. Typically 1% of the PSCA binding population was gated forcollection. Collected cells were grown in SD-CAA media and used for thenext round of sorting after induction in SG-CAA. Twenty yeast clonesfrom the fourth round of sorting were analyzed by flow cytometry. Eightof these clones showing strong staining were selected (A2, A4, A8, A9,A11, A12, B5 and C5) and their DNA sequenced. A2, A4, A8, A9, A12, B5protein sequences were identical with 10 mutations, A11 had 6 mutationsand C5 had 4 mutations. Protein sequence comparisons of the parental 2B3ScFv with A2, A11 and C5 are shown in FIG. 2.

Example 2

This example describes the reformatting of mutant 2B3 scFv's intominibodies.

The parental 2B3 minibody pEE12 construct (FIG. 2) was used as abackbone to generate the three 2B3 minibody affinity variants where thewild type ScFv insert was replaced by each of the three 2B3 ScFvaffinity variants. The parental 2B3 minibody construct is presented inFIG. 1. Briefly, V_(L) and V_(H) regions were fused with a 15 residuelong Gly-Ser rich linker in the V_(L) and V_(H) orientation. This ScFvis flanked by a signal peptide upstream and the human IgG1C_(H)3 domainvia the human IgGl hinge including a 10 residue GlySer peptide linkerdownstream. The final product was cloned into the PEE 12 vector ofexpression (Lonza Biologics, Slough, UK). This vector contains the hCMVpromoter and the glutamine synthetase gene for selection (Bebbington etal., Biotechnology (NY). 1992; 10: 169). The parental 2B3 minibody pEE12construct was used as a backbone to generate the three 2B3 minibodyaffinity variants. The parental pEE12 DNA was digested with Xba I andXho I restriction sites to remove the parental 2B3 ScFv insert that wasreplaced by each of the three 2B3 ScFv affinity variants. Xba I-Xho Irestriction sites were added at the extremities of 2B3 ScFv affinityvariants for sub-cloning in pEE12, by extension PCR.

Example 3

This example describes the expression, selection, and purification ofanti-PSCA minibodies.

A total of 2×10⁶ NSO mouse myeloma cells were transfected with 10 ug oflinearized (cut with SalI) vector DNA by electroporation and selected inglutamine-deficient media as described (Yazaki P J, et al. J ImmunolMethods. 2001; 253: 195). Clones were screened for expression by ELISA,whereby the desired protein was captured by goat anti-human IgG (Fespecific) and detected by alkaline phosphastase (AP)-conjugated goatanti-human IgG (Fe specific) (both from Jackson ImmunoResearch Labs,West Grove, Pa.). The highest producing clones were expanded and broughtto terminal culture.

Soluble minibodies were purified from cell culture supernatants byProtein L chromatography using a Thermal Separations Products HPLC withan in-line UV monitor, equipped with a preparative Poros 50 A column(Applied Biosystems, Foster City, Calif.) and analyzed on SDS-PAGE (FIG.3). The four minibodies showed similar results. All the minibodiesmigrated as molecular weight species of 95 kDa under non-reducingconditions and all showed good purity. In addition, the A11 minibodyeluted at 29.5 minutes as expected when run on a calibrated sizeexclusion column (FIG. 4). Supernatants were loaded onto a 10×50 mmcolumn and eluted using 0.1 M glycine pH 2.5, the pH was immediatelyneutralized with 2M Tris-HCl pH 8. The purified proteins were thendialyzed against PBS using a molecular porous membrane tubing (mwco:30,000) and concentrated with a Vivascience Vivaspin 20 (mwco: 30,000).Final protein concentrations were determined by measuring UV absorbanceat 280 nm, using the parental murine antibody as the standard.

Example 4

This example describes the biochemical characterization of anti-PSCAminibodies.

Size and composition: Purified proteins were analyzed by SDS-PAGE (FIG.3) under non-reducing conditions. Native structural size was determinedby size exclusion columns (Superdex 75) (Pharmacia).

The ranking of the four minibodies was determined by competition ELISAand flow cytometry (FIG. 5). The relative affinity as measured bycompetition ELISA indicated that all three affinity variants had higheraffinity than the parental, with an improvement of 4.4×, 3.0× and 1.9×for A2, A11 and C5 respectively compared to the parental. Flow cytometrydata also resulted in ranking the four minibodies in the same order whentargeting PSCA expressed at the cell surface. In conclusion, theaffinity ranking of the four minibodies was: A2>A11>C5>parental.

Competition ELISA: PSCA relative binding affinity for the minibodies wasdetermined by competition ELISA in which microtiter plate wells werecoated with purified PSCA-Fc (Olafsen T, et al. J. Irrununotherapy 2007:30:396).

Flow Cytometry: was conducted to assess cellular PSCA binding activity.An EBV transformed B-cell lymphoma cell line stably transfected withPSCA were used. Briefly, cells 5×10′ were incubated for 30 min on icewith 100 μl of minibody at 2 ug/ml concentration. Cells were washed andstained with goat anti-bFc PE conjugated antibody at 1:100 dilution.

Radioiodination: Purified minibodies were radioiodinated with thepositron emitting isotope ¹²⁴1 (sodium iodide in 0.02 M NaOH;radionuclide purity >99%) provided by Advanced Nuclide Technologies,Indianapolis, Ind. as previously described (Kenanova, Olafsen et al.,Cancer Res. 65:622, 2005). Immunoreactivity was assayed by incubatingradioiodinated-minibody with an excess amount of SKW-PSCA+ cells for anhour at room temperature, centrifugating the cells, and countingradioactivity present into the supernatant compared to the control.

Example 5

This example describes MicroPET imaging and biodistribution studies ofanti-PSCA minibodies.

All animal studies were conducted under protocols approved by theChancellor's Animal Research Committee at the University of California,Los Angeles. Xenografts were established in 7- to 8-week-old male nudemice (Charles River Laboratories, Wilmington, Mass.) by s.c. inoculationof 2×10⁶ LAPCP AD cells in the shoulder region. After 14 days, whentumor masses were in the range of 100 to 300 mg, 100 μCi of isotope ¹²⁴I(30-50μ protein) was injected into the tail vein of each animal. Micewere imaged using a P4 microPET scanner (Concorde Microsystems, Inc.,Knoxville, Tenn.). To enable imaging, mice were anesthetized using 2%isoflurane, positioned in a prone position along the long axis of themicroPET scanner and imaged. Acquisition time was 10 minutes (1 bedposition), and images were reconstructed using a filtered backprojectionreconstruction algorithm. Images were displayed and regions of interest(ROI) were drawn as described in FIG. 6 and quantified using AMIDE(Loening and Gambhir, Molecular Imaging 2: 13 1, 2003). After scanning,tumors, liver, spleen, kidney, lung, and blood were excised, weighed andcounted in a well counter (Cobra II AutoGamma, Packard, Ill.).Background, crossover, and decay corrections were done. Results werecalculated as percentage of injected dose per gram of tissue (% ID/g).

To evaluate tumor targeting and microPET imaging efficiency,¹²⁴1-labeled minibodies were injected into Nude mice bearing LAPC-9ADtumors on the right shoulder. Wholebody micoPET and CT scans wereperformed at 21 h and/or 25 h, after which the animals were sacrificed,and activity in various tissues quantified using a gamma counter. Toquantify microPET imaging, four 3-dimentionaJ RO is were drawn in thetumors and four other ROI in soft tissues around the tumor as presentedin FIG. 6. The ROI position and size were based on CT image information.Both biodistribution and imaging quantification are presented as a ratioof tumor signal to background. All minibody gave the bestbiodistribution and imaging data in an experiment that compared thethree affinity variant (FIG. 7A). Thus the affinity ranking (FIG. 5) andin vivo tumor targeting/imaging ranking (FIG. 7A) of the three affinityvariant minibodies are different, suggesting that in our model in vivotumor targeting/imaging effectiveness is not solely dependent on theinherent affinity of the tracer to its target. A2 which has the bestaffinity to PSCA did not give the best in vivo tumor targeting/imagingresults. One possible explanation for this discordance is that A2 has asubstitution of an asparagine into a tyrosine in VL CDR2, and thatiodination of this new tyrosine could affect the binding to PSCA. In asecond experiment A11 minibody was compared to the parental minibody fortheir in vivo tumor targeting/imaging effectiveness. A11 showed a 20%increase in tumor targeting (n=3) and a 141% increase in microPET tumorimaging (n=2) (FIG. 7B).

Example 6

This example describes the imaging of various pancreactic cancer tumorsusing anti-PSCA minibodies.

In order to evaluate the targeting and imaging potential of affinitymatured anti-PSCA minibodies, ¹²⁴I-labeled minibodies (parental 2B3 andvariant A11) were injected into athymic nude mice bearing tumors thatexpress low levels of target PSCA antigen. Briefly, xenographic micebearing either human Capan-1 (FIG. 14) or human MIA PaCa-2 (FIG. 15)pancreactic tumors were injected with either 200 or 300 μg of labeledanti-PSCA minibody. Wholebody micoPET and CT scans were performed asbefore, and tissue radioactivity was determined. Similarly,quantification of microPET imaging and ROI position and size weredetermined as in example 5.

As can be seen in FIGS. 14 and 15, variant A11 anti-PSCA microbodiesconsistently demonstrated improved tumor to muscle specificity ratios ascompared to parental 2B3 minibodies. The nearly 2-fold enhancement in invivo specificity suggests that these variant minibodies are bettersuited for use in therapeutic targeting and tumor imaging than existinganti-PSCA antibodies. Notably, tumor uptake in Capan-1 and HPAF-11 (datanot shown) was roughly 2% and less than 1% in MIA PaCa-2 tumors,suggesting specific uptake.

RELATED ART

-   1) Sundaresan, G., Yazaki, P. J., Shively, J. E., Finn, R. D.,    Larson, S. M., Raubitschek, A. A., Williams, L. E.,    Chatziioannou, A. F., Gambhir, S. S., and Wu, A. M. (2003)    Iodine-!24 labeled engineered anti-CEA minibodies and diabodies    allow highcontrast, antigen-specific small-animal PET imaging of    xenografts in athymic mice. J. Nucl. Med., 44:1962-1969.-   2) Olafsen, T., Gu, Z., Sherman, M. A., Leyton, J. V., Witkosky, M.    E., Shively, J. E.., Raubitschek, A. A., Morrison, S. L., Wu, A. M.    and Reiter, R. E. (2007) Targeting, imaging, and therapy using a    humanized anti-prostate stem cell antigen (PSCA) antibody. J.    lmmunotherapy 30:396-405.-   3) Leyton, J. V., Olafsen, T., Sherman, M. A., Reiter, R. E., and    Wu, A. M.-   Anti-prostate stem cell antigen (PSCA) antibody fragments for PET    imaging of prostate cancer (abstract). Cancer Biotherapy &    Radiopharmaceuticals 21:391, 2006.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the extent consistent with the presentdisclosure.

1-42. (canceled)
 43. An antigen binding protein construct that binds tohuman prostate stem cell antigen (PSCA) comprising an antigen bindingdomain comprising a heavy chain variable domain comprising thecomplementarity determining regions (CDRs) of the heavy chain variabledomain of SEQ ID NO: 6 and a light chain variable domain comprising theCDRs of the light chain variable domain of SEQ ID NO:
 6. 44. The antigenbinding protein construct of claim 43, wherein said antigen bindingdomain comprises a light chain variable domain comprising the lightchain variable domain of SEQ ID NO:
 6. 45. The antigen binding proteinconstruct of claim 43, wherein said antigen binding domain comprises aheavy chain variable domain comprising the heavy chain variable domainof SEQ ID NO:
 6. 46. The antigen binding protein construct of claim 43,wherein the construct is a minibody.
 47. The antigen binding proteinconstruct of claim 46, wherein said minibody comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:
 11. 48. Theantigen binding protein construct of claim 47, wherein said minibodycomprises the amino acid sequence of SEQ H) NO:
 11. 49. The antigenbinding protein construct of claim 43, wherein the construct is adiabody.
 50. The antigen binding protein construct of claim 43, whereinthe construct is a humanized antibody.
 51. The antigen binding proteinconstruct of claim 43, wherein the construct is an affinity maturedantibody.
 52. The antigen binding protein construct of claim 43, whereinthe construct is conjugated to a therapeutic agent.
 53. The antigenbinding protein construct of claim 52, wherein the therapeutic agent isa cytotoxic agent.
 54. The antigen binding protein construct of claim53, wherein the cytotoxic agent is selected from a group consisting ofricin, ricin A-chain, doxorubicin, daunotubicin, paclitaxel, ethidiumbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin,Pseudomonas, exotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin Achain, alpha-sarcin, gelonin rnitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheaniicin, sapaonaria officinalisinhibitor, maytansinoids, and glucocorticoidricin.
 55. The antigenbinding protein construct of claim 52, wherein the therapeutic agent isa radioactive isotope.
 56. The antigen binding protein construct ofclaim 55, wherein the radioactive isotope is ²¹²Bi, ¹³¹I, ¹¹¹In, ⁹⁰Y or¹⁸⁶Re.
 57. The antigen binding protein construct of claim 43, whereinthe construct is linked to an anti-cancer pro-drug activating enzymecapable of converting a pro-drug to its active form.
 58. The antigenbinding protein construct of claim 43, which is labeled with adetectable marker.
 59. The antigen binding protein construct of claim58, wherein the detectable marker is a radioisotope, a fluorescentcompound, a bioluminescent compound, a chemiluminescent compound, ametal chelator or an enzyme.
 60. The antigen binding protein constructof claim 58, wherein the detectable marker is ¹²⁴I, ⁸⁶Y, ¹⁸F, or ⁹⁴Tc.61. A pharmaceutical composition comprising the antigen binding proteinconstruct of claim 43 and a pharmaceutically acceptable excipient,carrier, or stabilizer.
 62. The composition of claim 61, which isformulated as an aqueous solution or a lyophilized solid.
 63. Apolynucleotide encoding the antigen binding protein construct of claim43.
 64. A method for inhibiting the growth of a prostate, pancreatic orbladder cancer cell expressing PSCA, said method comprising contactingsaid cancer cell with an antigen binding protein construct of claim 43,wherein said antigen binding protein construct is conjugated to aneffector moiety that is effective to inhibit the growth of said cancercell.
 65. The method of claim 64, further comprising administering tothe cancer cell a chemotherapeutic agent.
 66. The method of claim 64,further comprising administering radiation therapy to the cancer cell.67. A method for killing a prostate, pancreatic or bladder cancer cellexpressing PSCA, said method comprising contacting said cancer cell withan antigen binding protein construct of claim 43, wherein said antigenbinding protein construct is conjugated to an effector moiety that iseffective to kill said cancer cell.
 68. A method for treating aprostate, pancreatic or bladder cancer expressing PSCA in a patient,said method comprising administering to said patient an antigen bindingprotein construct of claim 43, wherein said antigen binding proteinconstruct is conjugated to an effector moiety that is effective toinhibit the growth of said cancer.
 69. The method of claim 68, furthercomprising administering to the patient hormone ablation therapy orhormone antagonist therapy.
 70. The method of claim 68, wherein theantigen binding protein construct is administered intravenously,intraperitoneally intramuscularly, intratumorally, or intradermally. 71.A method for imaging prostate, pancreatic or bladder cancer cellsexpressing MIA in a patient, said method comprising administering tosaid patient an antigen binding protein construct of claim 43, whereinsaid antigen binding protein construct is conjugated to a detectablemoiety, and detecting the presence and location of the antigen bindingprotein construct within the patient's body.
 72. The method of claim 71,wherein imaging is performed using a method selected from the groupconsisting of magnetic resonance imaging (MRI), nuclear magneticresonance (NMR), computerized axial tomography (CAT) scan, cooledcharged coupled device (CCD) camera optical imaging, bioluminescentoptical imaging, position emission tomography (PET), single photonemission computed tomography, and microPET.
 73. A method for detecting acancerous cell expressing PSCA, said method comprising contacting asample comprising cells with an antigen binding protein construct ofclaim 43 and detecting any complex of a cancerous cell expressing PSCAand said antigen binding protein construct that forms.
 74. An antibodythat binds to human prostate stem cell antigen (PSCA) comprising anantigen binding domain comprising: a heavy chain variable domaincomprising the complementarity determining regions (CDRs) of the heavychain variable domain of SEQ ID NO: 6; and a light chain variable domaincomprising the CDRs of the light chain variable domain of SEQ ID NO: 6.75. The antibody of claim 74, wherein said antibody is a minibody, adiabody, a scFv or a scFv-Fc.